Solvation and surface effects on polymorph stabilities at the nanoscale.

We explore the effects of particle size and solvent environment on the thermodynamic stability of two pairs of polymorphs subjected to ball-mill neat grinding (NG) and liquid assisted grinding (LAG). Two systems were studied: (i) forms I and II of a 1 : 1 theophylline : benzamide cocrystal and (ii) forms A and B of an aromatic disulfide compound. For both systems, the most stable-bulk polymorph converted to the metastable-bulk polymorph upon NG. LAG experiments yielded different outcomes depending on the amount of solvent used. This was further investigated by performing carefully controlled LAG experiments with increasing µL amounts of solvents of different nature. With these experiments, we were able to monitor form A to B and form I to II conversions as a function of solvent concentration and derive polymorph equilibrium curves. The concentration required for a switch in polymorphic outcome was found to be dependent on solvent nature. We propose that these experiments demonstrate a switch in thermodynamic stability of the polymorphs in the milling jar. Form B, the stable-bulk polymorph, has less stable surfaces than form A, thus becoming metastable at the nanoscale when surface effects become important. Ex situ diffraction and electron microscopy data confirm crystal sizes in the order of tens of nanometers after the ball mill grinding experiments reach equilibrium. DFT-d computations of the polymorph particles stabilities support these findings and were used to calculate cross-over sizes of forms A and B as a function of solvent. Attachment energies and surface stabilities of the various crystalline faces exposed were found to be very sensitive to the solvent environment. Our findings suggest that surface effects are significant in polymorphism at the nanoscale and that the outcomes of equilibrium ball-mill NG and LAG experiments are in general controlled by thermodynamics.

Coordination chemistry of Rh(III) porphyrins: complexes with hydrazine, disulfide, and diselenide bridging ligands.

Rh(III) porphyrin complexes with bridging hydrazine and substituted hydrazine ligands were characterized in solution by (1)H NMR spectroscopy and in the solid state by X-ray diffraction. Addition of further ligand to these species afforded 1:1 complexes in which methylhydrazine and N,N-dimethylhydrazine preferentially bound to the Rh center through the substituted nitrogen atom, as evidenced by (1)H NMR chemical shifts. An alkylated Rh(III) porphyrin was isolated as a decomposition product of the reaction of N,N-dimethylhydrazine with Rh(III) porphyrin in the presence of light and oxygen. Me(2)Se(2) and Me(2)S(2) formed bridging and nonbridging complexes with Rh(III) porphyrin, analogous to that observed with N,N'-dimethylhydrazine.

A strategy for the assembly of multiple porphyrin arrays based on the coordination chemistry of Ru-centered porphyrin pentamers.

An approach which employs pentameric porphyrin arrays as building blocks toward larger porphyrin arrays is described. Two flexible, and one relatively rigid, Ru-centered porphyrin pentamers (1-3) were synthesized and fully characterized. Their potential as building blocks toward larger porphyrin arrays has been studied via their coordination chemistry using bidentate and tetradentate ligands. DABCO (diazabicyclo[2.2.2]octane) can bind two monomeric porphyrins but was found to be too small to allow the complete formation of a 10-porphyrin array. On the other hand, titration of a larger bridging dipyridyl porphyrin ligand 17 (0.5 equiv) with 1 or 2 and tetrapyridyl ligand 18 (0.25 equiv) with 3 results in the formation of the 11-porphyrin and 21-porphyrin arrays, respectively, with the 21-porphyrin array containing porphyrins in three different metalation states. Changes in the chemical shift of the inner NH protons as well as the ortho- and meso-protons of the pyridyl groups of the porphyrin ligand clearly indicate the formation of large multiple porphyrin complexes. These studies demonstrate that by use of carefully designed building blocks and suitable bridging ligands, porphyrin arrays can be constructed with a dramatic increase in size in relatively few steps. Exploiting the fact that the strength of binding of pyridyl ligands is Ru > Zn > Ni, intra- vs intermolecular competition has been used to investigate aspects of the folding of the array. The photophysical properties of 3 are also described.

Rh(III) porphyrins as building blocks for porphyrin coordination arrays: from dimers to heterometallic undecamers.

The coordination chemistry of a Rh(III) porphyrin building block was investigated with a view to the construction of heterometallic arrays of porphyrins. The Rh(III) porphyrin was found to coordinate methanol in the solid state and weakly in CDCl(3) solution. Crystallization afforded five coordinate pi stacked Rh(III) porphyrins. The distribution of products from reaction of Rh(III) porphyrin with DABCO, 4,4'-bipyridine, and 4,4'-bipyrimidine could be displaced toward dimeric species by silica gel column chromatography or recrystallization which served to remove excess ligand. Weak coordination to nitriles was observed, although it was sufficiently strong to organize a dimeric complex of 5,5'-dicyano-2,2'-bipyridine in the solid state. Complexes with 4,4'-bipyrimidine and 5,5'-dicyano-2,2'-bipyridine possess uncoordinated chelating nitrogen atoms. Larger heterometallic porphyrin arrays were assembled using a combination of Sn(IV) and Rh(III) porphyrin coordination chemistry. A Sn(IV) porphyrin acted as a core around which were coordinated two isonicotinate groups, carboxylic acid functionalized porphyrins, or porphyrin trimer dendrons. Rh(III) porphyrins were coordinated to pyridyl groups at the periphery of these entities. In this way an eleven porphyrin array, with four different porphyrin metalation states, was assembled. The diamagnetic nature of both the Rh(III) and Sn(IV) porphyrins, the slow ligand exchange kinetics on the NMR time scale, and tight ligand binding permitted the porphyrin arrays to be analyzed by two-dimensional (1)H NMR techniques.

Structure-activity relationships in the acceleration of a hetero Diels-Alder reaction by metalloporphyrin hosts.

At 0.33 mM in dichloromethane at 25 degrees C the cyclic metalloporphyrin hosts 5, 7, 8, and 10 accelerate 12-fold, 260-fold, 1130-fold, and 250-fold, respectively, the reaction of 1 and 2 and also bind the product 3 very strongly. These observations combined with previously measured results with hosts 6, 9, and 11 allowed us to explore the influence of host geometry changes on acceleration rates and product binding over a wide range of host molecules of different size, ranging from extremely tightly strapped to very relaxed. To estimate the Zn-Zn distance in the transition-state complex, we carried out quantum mechanical calculations (at the HF/6-31G level) for the transition state to form 3. The structure-activity relationships found for hosts 5-11, along with the structural features calculated for the transition-state structure between 1 and 2 and previously crystallographically observed for product-free hosts and for a host.product complex,(4) suggest that both host preorganization as well as host flexibility are key features leading to high acceleration rates and product binding and that it is the delicate balance between the two structural features that leads to maximum efficiency.

Molecular evolution: dynamic combinatorial libraries, autocatalytic networks and the quest for molecular function.

The principles of Darwinian evolution have been explored in molecular systems such as autocatalytic networks and dynamic combinatorial libraries. Molecular evolution in such systems manifests itself as ligand or receptor amplification by selection. Research efforts exploring these concepts may provide a mechanism for the identification of novel catalysts, molecular receptors and bioactive molecules.

Electrospray ionisation Fourier-transform ion cyclotron resonance mass spectrometry of dynamic combinatorial libraries.

The use of electrospray ionisation Fourier-transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) for the analysis of dynamic combinatorial libraries (DCLs) of pseudo-peptide macrocyclic hydrazone oligomers is presented. The design of library building blocks results in mixtures of compounds with greater diversity than libraries generated by conventional combinatorial chemistry and so presents increased demands for analysis. The extended capabilities of the FTICR technique, specifically selective ion trapping, sensitivity, high resolution and mass accuracy over a broad mass range, are compatible with these increased demands and, most importantly, without the need for chromatography. Preliminary studies on the sequencing of cyclic oligomers and confirmation of the presence of sequence isomers are presented. These studies highlight the potential of FTICR-MS as a superior technique for the analysis of combinatorially generated compounds.

Synthesis and recognition properties of a ruthenium(II)-bis(zinc) cyclic porphyrin trimer.

A series of cyclic metalloporphyrin trimers containing one Ru(II)-CO porphyrin center are synthesized. A stepwise convergent route is used to synthesize Ru(CO)Zn(2)2.Py3T, where tripyridyltriazine (Py3T) templates the formation of the trimer and forces the CO group to the outside of the cavity. Three mixed-metal trimers, Ru(CO)Zn(2)2, Ru(CO)Ni(2)2, and Ru(CO)Mg(2)2, are synthesized from Ru(CO)Zn(2)2.Py3T and are characterized by NMR, UV-visible, and fluorescence spectroscopy. The Ru(CO)Zn(2)2 trimer is found to bind Py3T very tightly (K approximately 10(12) M-1), the resultant complex dissociating very slowly (kdissoc approximately 3 x 10(-7) s-1) in CDCl3 at 60 degrees C. During the course of these studies, the binding selectivity of a ruthenium porphyrin monomer, Ru(CO)3, for pyridine over THF is estimated to be ca. 7 x 10(4):1.

A tin(IV)-ruthenium(II)-tin(IV) cyclic porphyrin trimer with replaceable chiral linings.

A series of cyclic metalloporphyrin trimers, Ru(CO)(py)[Sn(carboxylate)2](2)2, having cavities lined with different carboxylate groups are synthesized by adding the appropriate carboxylic acids to the mixed trimer Ru(CO)(py)-[Sn(OH)2](2)2. This methodology provides ready access to a wide range of cavities lined with substituents possessing chirality or hydrogen-bonding groups. The potential of such systems is illustrated by synergistic binding of a hydroxypyridine to the Ru(II)-CO center and to the hydroxyl groups of a D-quinate-lined cavity. The effect of changing the carboxylate lining of the cavity on the course of epoxidation reactions catalyzed by these trimers is also reported.

Generation and screening of synthetic receptor libraries.

Efficient methods for the preparation of combinatorial libraries of receptors have been reported recently and, in most cases, these have led to the identification of host-like molecules that bind a particular substrate selectively. Dynamic libraries of macrocycles have also been synthesised that could open the way to a new selection approach to receptors and catalysts.

Enzyme models: design and selection.

There are two approaches to the discovery of enzyme mimics, that is identifying molecules that are able to bind substrate(s) and then catalyse reactions. The first approach, often inspired by enzymes themselves, utilises chemical knowledge and experience to design the catalyst. The other approach is to create a library and select the best host of a transition state analogue of the required reaction.

Vitamin D in infant formula and enteral products by liquid chromatography: collaborative study.

Results from a collaborative study of a new liquid chromatographic (LC) method for determination of vitamin D in infant formulas and enteral products are presented. Each of 15 laboratories was provided with 11 blind duplicate samples covering a range of approximately 200-500 International Units/quart (normal dilution), a system suitability sample, and the U.S. Pharmacopeia ergo- and cholecalciferol standards. Product types included liquid and powder forms of milk (whey and casein), soy, and hydrolyzed protein-based infant formulas and enteral products. The method includes a single liquid-liquid extraction following saponification, solid-phase extraction, and then concentration by evaporation. An isocratic, nonaqueous, chromatographic system with reversed-phase, zero endcapped C18 column, and UV detector set at 265 nm are used. Statistical evaluation of data from participating laboratories show the average reproducibility and repeatability of the method across all samples to be excellent, with RSDR and RSDr values of 13.48 and 9.44, respectively, after elimination of outliers. The LC method for determination of vitamin D in infant formulas and enteral products has been adopted by AOAC INTERNATIONAL.

NMR study of whole rat bile: the biliary excretion of cefoperazone and benzyl chloride by an isolated perfused rat liver.

1H NMR spectroscopy at 400 MHz has been applied to the analysis of whole bile samples produced by the isolated perfused rat liver. Using relatively simple NMR experiments biliary excretory products of cefoperazone and benzyl chloride were identified as cefoperazone itself and a benzyl-glutathione conjugate, respectively. Our use of 13C isotopic labelling demonstrates how 1H/13C heteronuclear NMR techniques can be used to produce uncrowded and informative spectra from whole bile. From the use of a HMQC-COSY experiment the structure of a benzyl-glutathione conjugate contained in whole bile was confirmed.

NMR study of whole rat bile: the biliary excretion of 4-cyano-N,N-dimethyl aniline by an isolated perfused rat liver and a liver in situ.

The structure of two biliary metabolites of 4-cyano-N,N-dimethyl aniline (CDA) contained in whole rat bile have been studied in detail by NMR at 400 MHz. A 4-cyano-N-methyl glutathione-N-aniline conjugate was identified as a biliary metabolite of CDA using relatively simple 1H NMR techniques. Isotopically 13C labelled CDA was used to generate 13C labelled xenobiotic conjugates. Our use of 1H/13C heteronuclear NMR techniques, in particular a 13C-selective HMQC-TOCSY experiment, allowed a N-beta-glucuronide conjugate, a previously unknown biliary metabolite of CDA, to be identified. Bile samples obtained from both the isolated perfused rat liver and the rat liver in situ were analysed.

Nuclear magnetic resonance relaxation studies of poly(hydroxybutyrate) in whole cells and in artificial granules.

The physical state of poly(hydroxybutyrate) (PHB) in whole cells and in the form of artificial biomimetic granules has been probed using 13C nuclear magnetic resonance (NMR) spectroscopy. Studies on varying concentrations of whole cells of Alcaligenes eutrophus show that changes in the line widths of PHB in whole cells do not correlate with changes in transverse relaxation times. Solid-state magic-angle spinning NMR studies demonstrate that the line broadening results from a reduction in the static field homogeneity rather than from intrinsic properties of the PHB within the cells. Transverse and longitudinal relaxation times of PHB in whole cells and in artificial granules are similar, indicating similarities in structure and mobility.

Male antifertility compounds from Tripterygium wilfordii Hook f.

Extracts of the Chinese medicinal plant, Tripterygium wilfordii, cause reversible infertility in male animals. Sub-fractionation studies have now revealed that the plant extracts contain a number of compounds which are potent antifertility agents in male mammals, including the diterpenes triptolide and tripdiolide and an isomer of the latter. A triptolide, 12,13-chlorohydrin, which is a transformation product formed reversibly by interaction of triptolide with HCl, was also found to be active.

Plasticization of poly(hydroxybutyrate) in vivo.

The influence of a variety of treatments on the mobility and crystallinity of poly(hydroxybutyrate) (PHB) in whole cells and native granules has been proved using 13C-n.m.r. spectroscopy and X-ray powder diffraction, and correlated with the known biological effects of these treatments. It was concluded that at least water is responsible for PHB plasticization in vivo, and that only native mobile PHB is susceptible to depolymerases. Another, probably hydrophobic, component appears to be involved either as plasticizer or nucleation inhibitor. Three states of the granule are identified in addition to the native, biologically-competent state: freeze-drying of whole cells leads to a partially-immobilized amorphous state which can be restored virtually to native mobility by rehydration; extended centrifugation of native granules in aqueous suspension, or treatment with hydrophobic detergents under certain conditions, leads to a crystalline state that is less susceptible to exogenous depolymerase; and heating to 95 degrees C or refrigeration has no detectable effect on mobility but leads to inactivation of the granule, presumably via damage to superficial membrane or protein.

A reverse approach to 1H-n.m.r. assignments of bacterial polysaccharides.

A new approach is reported for obtaining the assignment of the 1H-n.m.r. spectra of bacterial polysaccharides which are not amenable to analysis using conventional strategies. An unambiguous assignment of the 13C-n.m.r. spectrum is made by 13C-COSY of the polysaccharide labelled to a high level with 13C. Assignment of the 1H-n.m.r. spectrum is then made by proton-detected CH-correlation spectroscopy of material that is labelled at a low level. Using this approach, virtually complete assignments have been made for the Klebsiella K3 serotype polysaccharide under physiological conditions of temperature and pH.

Glutathione-dependent dechlorination of 1,6-dichloro-1,6-dideoxyfructose.

The metabolism of 14C- and 36Cl-labelled 1,6-dichloro-1,6-dideoxyfructose (DCF) was studied in the isolated perfused rat liver system. Dechlorination of DCF occurred in the liver and erythrocytes and was GSH-dependent. The GSH conjugate formed was identified by 13C and 1H n.m.r. as the 6-chlorofructos-1-yl-SG conjugate. It is proposed that the GS- anion attacks the low steady-state concentration of the reactive keto form of DCF and that the conjugate formed cyclizes to the dominant beta-anomer. 6-Chlorofructos-1-yl-SG conjugate of hepatic origin is excreted into bile, whereas that produced in erythrocytes does not enter the liver.